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    Chinese Journal of Chromatography
    2017, Vol. 35, No. 9
    Online: 08 September 2017

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    A novel ship-borne positive pressure solid phase extraction device to enrich organo chlorinated and pyrethroid pesticides in seawater
    YE Jianglei
    2017, 35 (9):  907-911.  DOI: 10.3724/SP.J.1123.2017.05013
    Abstract ( 548 )   [Full Text(HTML)] () PDF (1227KB) ( 93 )  

    A novel solid phase extraction (SPE) device driven by positive pressure was developed instead of negative pressure from a vacuum pump, in order to enrich organo chlorinated and pyrethroid pesticides in seawater. The water sampling bottles and the pipelines which touch water samples were made of plastic material without chlorine. In order to ensure the sealing and firmness, the whole device were tightened with nut and bolt. The inner pressure (0.1-0.3 MPa) in the water sampling bottle was provided by the small air pump (powered by 12 V cell) controlled by a microprogrammed control unit (MCU) and pressure sensor to keep the water flow rate (4.0-6.0 mL/min). The pre-conditioned SPE column can be used for the enrichment of pesticides within four weeks, and the loaded SPE column can be eluted for detection within six weeks with recoveries greater than 80%. The linearity of the method was good with the correlation coefficient more than 0.9. The limits of quantification (LOQs) were 0.8-6 ng/L. The recoveries of the pesticides at three spiked levels (3 parallel samples) were 86.1%-95.5% with the relative standard deviations less than 10%. The benzene hexachlorides (BHCs) and dichloro-diphenyl-trichloroethanes (DDTs) were detected in seawater samples. The device has good application in enriching organo chlorinated and pyrethroid pesticides in seawater.

    Preparation of filter paper with chiral separation function by oxidation and Schiff-base reaction
    LUO Lixin, ZHAO Xinghua, JIANG Minjie, ZHANG Haiyan, WANG Yu
    2017, 35 (9):  912-917.  DOI: 10.3724/SP.J.1123.2017.04020
    Abstract ( 455 )   [Full Text(HTML)] () PDF (1032KB) ( 97 )  

    Paper chromatography (PC) is a highly flexible, fast, and efficient separation method. In this study, a chromatographic paper with a chiral separation function was investigated and a paper chromatographic method for chiral separation was developed. Single-factor experiments and orthogonal tests were used to determine the optimal oxidation conditions for filter paper. The optimal conditions were that sodium periodate 4% (mass percentage), pH 2 buffer solution, reaction temperature 45℃, and reaction time 4 h. Under the optimal conditions, the aldehyde group content of a dialdehyde-based filter paper was 57.93% (amount of substance percentage). A paper-based chiral separation material was synthesized by a microwave-assisted Schiff-base reaction of the oxidized filter paper with L-glutamic acid. The developing solvent for separating racemic tartaric acid using this chiral filter paper consisted of 100 mL of n-butanol, 50 mL of 50%(v/v) acetic acid, and 0.1000 g of bromophenol blue. The rate of flow (Rf) values of L-tartaric acid and D-tartaric acid were 0.52 and 0.40, respectively. This method does not require large-scale equipment to perform the chiral separation and it is therefore suitable for general teaching, research, and industrial applications.

    Present situation and development trends of asymmetrical flow field-flow fractionation
    LIANG Qihui, WU Di, QIU Bailing, HAN Nanyin
    2017, 35 (9):  918-926.  DOI: 10.3724/SP.J.1123.2017.05021
    Abstract ( 810 )   [Full Text(HTML)] () PDF (1376KB) ( 166 )  

    Field-flow fractionation (FFF) is a kind of mature separation technologies in the field of bioanalysis, feasible of separating analytes with the differences of certain physical and chemical properties by the combination effects of two orthogonal force fields (flow field and external force field). Asymmetrical flow field-flow fractionation (AF4) is a vital subvariant of FFF, which applying a vertical flow field as the second dimension force field. The separation in AF4 opening channel is carried out by any composition carrier fluid, universally and effectively used in separation of bioparticles and biopolymers due to the non-invasivity feature. Herein, bio-analytes are held in bio-friendly environment and easily sterilized without using degrading carrier fluid which is conducive to maintain natural conformation. In this review, FFF and AF4 principles are briefly described, and some classical and emerging applications and developments in the bioanalytical fields are concisely introduced and tabled. Also, special focus is given to the hyphenation of AF4 with highly specific, sensitive detection technologies.

    Preparation and evaluation of hydrophilic interaction/reversed-phase mixed-mode chromatographic packing based on polylsilsesquioxane microspheres
    LÜ Qiannan
    2017, 35 (9):  927-933.  DOI: 10.3724/SP.J.1123.2017.05006
    Abstract ( 635 )   [Full Text(HTML)] () PDF (2315KB) ( 165 )  

    Hydrophilic interaction (HILIC)/reversed-phase (RPLC) mixed-mode chromatography is widely used in separating both hydrophobic and hydrophilic compounds, but its pH range is limited which is harmful to the separation of alkaline drugs. Monodispersed and porous cysteine-modified vinyl functionalized polymethylsilsesquioxane (C-V-PMSQ) microspheres were prepared by thiol-ene click chemistry. Elemental analysis revealed that cysteine was successfully bonded to the surface of microspheres. C-V-PMSQ microspheres had excellent monodispersity, favorable chemical stability and simple preparation process. The chromatographic behavior of C-V-PMSQ stationary phase was investigated by employing several nucleosides and nucleic acids under HILIC mode and RPLC mode. Retention factors versus volume percentage of aqueous solution exhibited a U-curve, which can be evaluated as an indication for HILIC/RPLC mixed-mode behavior of the stationary phase. This new stationary phase presented stronger retention behavior when employing alkylbenzenes as evaluation system. In addition, a series of hydrophilic and hydrophobic compounds were separated at the same time using this new stationary phase. Furthermore, baseline separation for the major three active components of Chinese herbal medicine Radix Sophorae flavescentis was achieved under HILIC and RPLC modes with highly alkaline mobile phase. The excellent chemical stability and base deactivated nature make the organosilicas ideal stationary phases for the separation of basic compounds. What's more, it even can achieve two-dimensional liquid chromatography separation on an HPLC column.

    Stable isotope labeling and parallel reaction monitoring-based proteomic quantification for biomarker screening and validation of hepatocellular carcinoma
    WANG Sulan, GAO Huaping, ZHANG Jing, YE Xiang
    2017, 35 (9):  934-940.  DOI: 10.3724/SP.J.1123.2017.05022
    Abstract ( 512 )   [Full Text(HTML)] () PDF (4165KB) ( 237 )  

    Liver cancer is the fifth most common cancer with extremely low five year survival rate. Early diagnosis is of great importance for cancer therapy. In this work, stable isotope labeling-based relative quantitative proteomics and parallel reaction monitoring-based target proteomics were combined for cancer biomarker screening and validation. By using this strategy, 70 significantly changed proteins in hepatocellular carcinoma tissues were obtained, among which seven proteins were further validated. The validated proteins contain the clinically used hepatocellular carcinoma (HCC) biomarker alpha-fetoprotein (AFP) and the reported biomarker candidates Heat shock protein HSP 90-beta (HSP90), fatty acid-binding protein, epidermal (FABP5) and alcohol dehydrogenase 4 (ADH4), which demonstrated the robustness of the strategy. The proteins identified in this work could be benefit for further HCC biomarker screening and clinical validation. Moreover, this strategy could be further applied to other cancer types.

    Rapid screening of 44 pesticide residues in ginger and scallion by ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry
    LIU Yongqiang, LIU Sheng, XU Wenjuan, TIAN Guoning, ZHANG Jinling
    2017, 35 (9):  941-948.  DOI: 10.3724/SP.J.1123.2017.06001
    Abstract ( 835 )   [Full Text(HTML)] () PDF (883KB) ( 123 )  

    An analytical method was established for the simultaneous determination of 44 pesticide residues in ginger and scallion by ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS). The samples were extracted with acetonitrile containing 0.1% (v/v) acetic acid aqueous solution, and cleaned-up by primary secondary amine (PSA) and octadecyl bonded silica (C18) adsorbents. The compounds were separated on a Poroshell 120 SB-C18 column (100 mm×3.0 mm, 2.7 μm) with 0.1% (v/v) formic acid aqueous solution containing 5 mmol/L ammonium acetate-acetonitrile as mobile phases under gradient elution. The eluent was determined by UPLC-Q-TOF/MS with electrospray ionization in positive mode. The quantification analysis was performed with the external standard method. In all ions MS/MS mode, the compounds were qualitatively screened and confirmed by one data acquisition. The correlation coefficients (r) were greater than 0.995 in the linear ranges of the 44 pesticides. The limits of quantification (LOQs, S/N=10) of the 44 pesticides were 2.5-5.0 μg/kg. At the three spiked levels, the recoveries were between 73.4% and 113.7% with the relative standard deviations (RSDs) ranging from 0.7% to 12.1% (n=6). The method effectively improves the determination efficiency of pesticide residues screening by high-resolution mass spectrometry.

    Rapid determination of 15 nutrients in rice by ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry
    ZHU Li, TAN Wei, PENG Zumao, DENG Mengya, ZHANG Xieguang, YANG Guowu
    2017, 35 (9):  949-956.  DOI: 10.3724/SP.J.1123.2017.05001
    Abstract ( 531 )   [Full Text(HTML)] () PDF (2405KB) ( 88 )  

    A rapid method was developed for the simultaneous determination of 15 nutrients containing eight vitamin E, six γ-oryzanols and β-carotene in rice by ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (UHPLC-LTQ/Orbitrap HRMS). The analytes were extracted by methanol containing 0.05% (v/v) 2,6-di-tert-butyl-4-methylphenol (BHT) under the ultrasonic condition, then separated by a Poroshell 120 PFP column (150 mm×3.0 mm, 2.7 μm) with a gradient elution program using 0.1% (v/v) formic acid aqueous solution and methanol containing 0.1% (v/v) formic acid as mobile phases. The detection was performed using an LTQ/Orbitrap HRMS detector with full scan in positive ion mode. Fifteen nutrients were simultaneously separated within 13 min. Furthermore, matrix effects of the white and brown rices were investigated. The correlation coefficients (r) were ≥ 0.9950 in the linear ranges of the 15 nutrients. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) of the 15 nutrients were 0.2-1.8 μg/L and 0.7-6.1 μg/L, respectively. At the three spiked levels, the recoveries were 73.2%-101.5% with the relative standard deviations (RSDs) ranging from 1.1% to 5.0% (n=3). The method is accurate, efficient and reliable. It is suitable for the simultaneous determination of various nutrients in rice.

    Rapid screening and identification of food poisonings by ultra high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry
    ZHU Feng, JI Wenliang, LIU Hualiang, JIA Yanbo, CAI Mei, ZHANG Hao
    2017, 35 (9):  957-962.  DOI: 10.3724/SP.J.1123.2017.04036
    Abstract ( 687 )   [Full Text(HTML)] () PDF (843KB) ( 176 )  

    A method was developed for the screening and detection of food poisonings by ultra high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF MS). After extracted by acetonitrile and cleaned-up by QuEChERS, the extract was separated on a Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) with the gradient elution of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile. TOF-MS scan-information dependent acquisition (IDA)-product ion scan was performed in positive electrospray ionization (ESI) mode to acquire high resolution MS and MS/MS spectra in one injection, and rapidly screen 581 target compounds by SCIEX OS software, including 546 pesticides, 24 mycotoxins, 11 rodenticides. The target compounds were qualitatively confirmed by mass accuracy of precursor, isotope distribution of precursor, fragment ions of precursor, and library search. Carbofuran was detected in 9 out of 11 samples with the proposed method. The retention time was further confirmed by the standard of carbofuran. The results showed the retention times were coincident between the samples and reference standard, and the deviations of accurate mass numbers were all less than 3.7×10-6. The scope of the relationship was good and the correlation coefficient was 0.998. The instrumental limit of detection (S/N=3) was 0.3 μg/kg, and the limit of quantification (S/N=10) was 1 μg/kg. The recoveries at 10, 50, 200 μg/kg levels were 75.6%-95.9%, and the RSDs (n=6) were 3.6%-6.9%. The method is rapid, simple, accurate and sensitive. It is suitable for the rapid screening and detection of public safety incidents.

    Determination of the plant growth regulators in batatas by matrix solid-phase dispersive extraction and high performance liquid chromatography with tandem mass spectrometry
    ZHANG Jing, WAN Huihui, ZHANG Hua
    2017, 35 (9):  963-969.  DOI: 10.3724/SP.J.1123.2017.06005
    Abstract ( 656 )   [Full Text(HTML)] () PDF (1323KB) ( 84 )  

    An analytical method based on matrix solid-phase dispersive (MSPD) extraction and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the determination of forchlorfenuron, 6-benzylaminopurine (6-BA), N-(2-ethylhexyl)-5-norbornene-2,3-dicarboximide (MGK 264) and paclobutrazol in batatas. The samples were dispersed with silica by grinding, and then extracted with methanol. The determination was carried out on a Thermo hypersil GOLD C18 column (150 mm×2.1 mm, 5 μm) in gradient elution with mobile phases of methanol and ammonium formate (5 mmol/L, containing 0.1% (v/v) formic acid) and detected with tandem mass spectrometry using selected reaction monitoring (SRM) mode. The quantification was performed using external standard calibration, and the calibration curves were performed in the ranges of 10.8-216.0 ng/g (forchlorfenuron), 10.8-216.0 ng/g (6-BA), 12.5-250.0 ng/g (MGK 264), 10.2-204.0 ng/g (paclobutrazol) with the correlation coefficients larger than 0.96. The limits of quantification (LOQs) were 0.1-0.3 ng/g. The spiked recoveries at the levels of 50, 100 and 200 ng/g of these four plant growth regulators were in the range of 85.3%-116.0%, and the relative standard deviations were 0.6%-22.7%. This method has advantages of simple operation and good accuracy, and can be used for the quantitative determination of forchlorfenuron, 6-BA, MGK 264 and paclobutrazol in batatas.

    Determination of strobilurin fungicides in fruits and their mass fragmentation routes by ultra performance liquid chromatography-tandem mass spectrometry
    ZHOU Yao, YANG Huiqin, SHI Yiyin, CHEN Jiaxian, ZHU Jian, DENG Xiaojun, GUO Dehua
    2017, 35 (9):  970-979.  DOI: 10.3724/SP.J.1123.2017.05009
    Abstract ( 699 )   [Full Text(HTML)] () PDF (1294KB) ( 127 )  

    A method was developed for the simultaneous determination of six strobilurin fungicide (E-metominostrobin, azoxystrobin, kresoxim-methyl, picoxystrobin, pyraclostrobin and trifloxystrobin) residues in orange, banana, apple and pineapple samples by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The fragmentation routes of all the compounds were explained by the aid of a fragment predicting software ACD Lab/MS Fragmenter. The samples were extracted by acetonitrile, then cleaned up by amino solid phase extraction cartridges (SupelClean LC-NH2). The extracts were separated on a ACQUITY UPLC BEH C18 column (50 mm×2.1 mm, 1.7 μm) with gradient elution. Acetonitrile containing 0.1% (v/v) formic acid and 10 mmol/L ammonium acetate containing 0.1% (v/v) formic acid were used as mobile phases. The samples were detected by electrospray ionization (ESI)-MS/MS in positive ion and multiple reaction monitoring (MRM) mode, quantified by external standard method. Good linearities were obtained in the range of 5-100 μg/L (for pyraclostrobin, 1-20 μg/L) with correlation coefficients (r2) greater than 0.999. The recoveries ranged from 60.4% to 120% with the relative standard deviations between 2.15% and 15.1% (n=6). The developed method can meet the inspection of the six strobilurin residues in the orange, banana, apple and pineapple samples.

    Rapid determination of four phenolic environmental estrogen residues in cooking oil by ultra-performance liquid chromatography-high resolution mass spectrometry coupled with solid-phase extraction
    PAN Shengdong, HE Qian, CHEN Xiaohong, WANG Li, QIU Qiaoli, JIN Micong
    2017, 35 (9):  980-986.  DOI: 10.3724/SP.J.1123.2017.06003
    Abstract ( 705 )   [Full Text(HTML)] () PDF (992KB) ( 92 )  

    A fast, sensitive and accurate method for the determination of trace bisphenol S (BPS), bisphenol F (BPF), bisphenol A (BPA) and 4-nonylphenol (4-NP) in cooking oil samples was developed by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) coupled with solid-phase extraction (SPE). Cooking oil samples were extracted by acetonitrile, then the supernatant was purified by SLC SPE cartridges. The chromatographic separation was carried out on a Waters ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) with a linear gradient elution procedure using 0.05% (v/v) triethanolamine aqueous solution and methanol as mobile phases. The quantification analysis was operated in a negative electrospray ion (ESI-) source mode under the selected ion monitoring (SIM) mode with internal standard method. The four target analytes showed good linearity with correlation coefficients (r) greater than 0.999. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) were in the ranges of 0.03-0.11 μg/kg and 0.10-0.36 μg/kg, respectively. The recoveries of the four target analytes spiked in oil samples were in the range of 86.3%-96.1% at spiked levels of 1.0, 10.0 and 80.0 μg/kg, respectively, while the relative standard deviations (RSDs) were in range of 2.2%-8.8% (n=6). No significant matrix interference was found in this method. The proposed method is simple and fast. It can be applied for the rapid determination of trace BPS, BPF, BPA, and 4-NP in cooking oil samples.

    Identification of the chemical compositionsof Verbena officinalis L. extract by high performance liquid chromatography-photodiode array-high resolution mass spectrometry
    SUN Yuming, WANG Yueyue, CAI Rui, ZHANG Hua, WANG Yulin
    2017, 35 (9):  987-994.  DOI: 10.3724/SP.J.1123.2017.05010
    Abstract ( 766 )   [Full Text(HTML)] () PDF (986KB) ( 167 )  

    To investigate the main chemical compositions of Verbena officinals L. extract, a qualitative high performance liquid chromatography-photodiode array-high resolution mass spectrometry (HPLC-PDA-HRMS) method was established. The structures of the compounds detected were identified by analyzing the chromatographic profiles and the corresponding mass spectra obtained by full scan and MSn full scan. Twenty one compounds including iridoid glycosides, flavonoids, triterpenoids, phenylpropanoids and phenolic diterpenoids were identified, and six of them were not reported in other literatures about Verbena officinalis L. extract, such as carnosic acid, carnosol, rosmanol, isorosmanol, rosmarinic acid and acacetin-7-O-rutinoside. This method is simple, rapid, accurate and sensitive. It provides a reliable scientific basis for the identification of the authenticity and quality control of Chinese medicinal materials.

    Simultaneous determination of diminazene aceturate and isometamidium chloride residues in cattle tissues by high performance liquid chromatography with solid-phase extraction
    ZHOU Yuhui, ZENG Zhenling, YU Jingxian, LIU Rong, PAN Lihua, ZHONG Qiuyu, CHEN Hong
    2017, 35 (9):  995-1002.  DOI: 10.3724/SP.J.1123.2017.04033
    Abstract ( 747 )   [Full Text(HTML)] () PDF (1391KB) ( 159 )  

    A method for the simultaneous determination of trypanocidal diminazene aceturate (DIM) and isometamidium chloride (ISM) that containing benzamidine groups in cattle tissues was developed by high performance liquid chromatography (HPLC) with solid-phase extraction (SPE). The tissue samples were extracted with different proportions of water-acetonitrile, then were cleaned up by Oasis WCX cartridges. DIM and ISM were separated by HPLC with a Spherisorb CN column (250 mm×4.6 mm, 5 μm). Acetonitrile-0.05 mol/L ammonium formate solution (pH 2.4) was used as mobile phases with gradient elution. The detection wavelength of UV was set at 380 nm. The limits of detection (LODs) and the limits of quantification (LOQs) of DIM and ISM in cattle tissues were 0.01 mg/kg and 0.025 mg/kg, respectively. The correlation coefficients (r) of DIM and ISM in cattle tissues were not less than 0.9993. The average recoveries of DIM and ISM at three spiked levels were 82.2%-97.6% with the intra-day relative standard derivations (RSDs) of 0.3%-5.2% (n=5) and inter-day RSDs of 1.3%-5.2% (n=15). The method was successfully applied to the analysis of DIM and ISM in cattle tissues. The method is rapid, sensitive and repeatable for the determination of diminazene aceturate and isometamidium chloride in cattle tissues.

    Supramolecular solvent-based direct extraction for the determination of polycyclic aromatic hydrocarbons in water
    TAN Yimeng, TANG Tao, YANG Sandong, XIA Mingzhu, WANG Fengyun, LI Tong
    2017, 35 (9):  1003-1007.  DOI: 10.3724/SP.J.1123.2017.06004
    Abstract ( 561 )   [Full Text(HTML)] () PDF (1109KB) ( 76 )  

    Supramolecular solvent (SUPRAS) is a nano-structured liquid generated from amphiphiles through a sequential self-assembly process. It is an efficient and excellent solvent for the sample extraction. In this paper, a method to directly extract and rapidly analyze polycyclic aromatic hydrocarbons (PAHs) in water samples by high performance liquid chromatography-fluorescence detection (HPLC-FLD) was developed. The composition and amount of SUPRAS were optimized and practical samples were tested. It indicated that the combination of tetrahydrofuran and 1-octanol was a suitable SUPRAS for the extraction of four PAHs with recoveries between 89.08% and 102.47% and relative standard deviations (RSDs) from 1.38% to 3.92% (n=5). Results showed a good linearity of four PAHs with the correlation coefficients (R2) more than 0.999. The limits of detection (LODs) ranged from 1.26 to 9.23 ng/L. The proposed pretreatment method greatly reduces the analysis time. And the solvent-less approach is in accordance with the development trend of green chemistry and of great application prospects.

    Determination and prediction for vapor pressures of organophosphate flame retardants by gas chromatography
    WANG Qingzhi, ZHAO Hongxia, WANG Yan, XIE Qing, CHEN Jingwen, QUAN Xie
    2017, 35 (9):  1008-1013.  DOI: 10.3724/SP.J.1123.2017.05015
    Abstract ( 641 )   [Full Text(HTML)] () PDF (848KB) ( 95 )  

    Organophosphate flame retardants (OPFRs) are ubiquitous in the environment. To better understand and predict their environmental transport and fate, well-defined physicochemical properties are required. Vapor pressures (P) of 14 OPFRs were estimated as a function of temperature (T) by gas chromatography (GC), while 1,1,1-trichioro-2,2-bis (4-chlorophenyl) ethane (p,p'-DDT) was acted as a reference substance. Their log PGC values and internal energies of phase transfer (△ vapH) ranged from -6.17 to -1.25 and 74.1 kJ/mol to 122 kJ/mol, respectively. Substitution pattern and molar volume (VM) were found to be capable of influencing log PGC values of the OPFRs. The halogenated alkyl-OPFRs had lower log PGC values than aryl-or alkyl-OPFRs. The bigger the molar volume was, the smaller the log PGC value was. In addition, a quantitative structure-property relationship (QSPR) model of log PGC versus different relative retention times (RRTs) was developed with a high cross-validated value (Q2cum) of 0.946, indicating a good predictive ability and stability. Therefore, the log PGC values of the OPFRs without standard substance can be predicted by using their RRTs on different GC columns.

    Separation and purification of acteoside from Rehmannia glutinosa by combining macroporous resin with high-speed countercurrent chromatography
    BU Zhisi, HE Qing, ZHAO Rushi, CHU Chu, LI Xingnuo, TONG Shengqiang
    2017, 35 (9):  1014-1021.  DOI: 10.3724/SP.J.1123.2017.04030
    Abstract ( 619 )   [Full Text(HTML)] () PDF (905KB) ( 106 )  

    Acteoside,a chemical component of the traditional Chinese medicinal herb Rehmannia glutinosa,was separated from the plant by a combination of macroporous resin column chromatography and high-speed countercurrent chromatography.The static adsorption and desorption of acteoside in the crude extract were in-vestigated using four kinds of macroporous resin,among which the D101 macroporous resin presented the best adsorption and desorption rates toward the compound.The crude extract was then gradient-eluted with increa-sing volume percentages of ethanol,where it was found that the highest content of acteoside was obtained when using 10%(v/v) ethanol eluent,in which the purity of acteoside was increased from 4.9% to 32.6%.Then,the partially purified crude extract (165 mg) was further purified by high-speed countercurrent chroma-tography using a two-phase solvent system consisting of ethyl acetate-n-butanol-water (1:4:5,v/v/v),yielding 45 mg of acteoside with 96% purity.